For cancer treatment, various treatment methods, including surgery, radiotherapy and chemotherapy, have been developed and used. However, in the case of particular cancers and patients, these treatment methods are difficult to apply, and there is a high possibility of recurrence.
Thus, immunotherapy utilizing the immune function of patients has received increasing attention and is based on the removal of cancers by the complex interactions between immune cells having various functions. Immune cells that directly remove cancer cells include NK cells and cytotoxic T lymphocytes (CTLs), and that present antigens to these effector cells include dendritic cells (DCs) or B cells. Other examples include helper T cells (Th cells), regulatory T cells (Treg cells) and the like, which secrete various cytokines. Among these immune cells, NK cells are recognized as the most rapidly effective and efficient immune cells.
NK cells are lymphocytes that account for about 10% of blood cells and play an important role in immune responses. NK cells have various functions, and particularly have the ability to kill cancer cells or cells infected with external pathogenic bacteria, and thus function to remove cancer cells, or cells developing into cancers.
Most NK cells are normally present in the body in an inactivated state, but in order to use NK cells for therapeutic purposes, NK cells need to be activated. Thus, studies on the activation of NK cells from normal blood or the inactivated blood of patients have been actively conducted.
The high cytotoxicity of NK cells, achieved by activating NK cells ex vivo, demonstrated the possibility of NK cells for immune cell therapy. It was reported that NK cells activated ex vivo have the rapeutic effects on various cancers, particularly blood cancer such as leukemia, when they are administered after allogenic bone marrow transplantation (Blood Cells Molecules & Disease, 33: p 261-266, 2004). However, the distinct therapeutic effects of NK cells on solid cancers other than blood cancer have not yet been clinically proven. Specifically, it was reported that the administration of NK cells before the formation of cancers can interfere with the engraftment of cancers (Cancer Immunol. Immunother., 56(11): p 1733-1742, 2007), but this treatment model does not appear to be suitable. In addition, it was reported that intraperitoneal administration of NK cells in animal tests inhibited the growth of breast cells, but it is unclear whether this inhibitory effect is attributable to NK cells (Breast Cancer Res. Treatment, 104(3): p 267-275, 2007).
Meanwhile, despite the possibility of NK cells as therapeutic agents for cancers or infectious diseases, the number of NK cells present in vivo is not large, and thus there is required a technology of producing large amounts of NK cells while maintaining efficiency sufficient for therapeutic purposes. However, NK cells are not sufficiently cultured and expanded in vitro. Thus, a technology for culturing and expanding NK cells at useful levels has received attention, and many studies thereon have been conducted, but the study results are still not clinically applicable.
There have been studies on the culture of NK cells not only using IL-2, which has been in T-cell proliferation/activity, but also IL-15 (J. Immunol., 167(6): p 3129-3138, 2001; Blood, 106(1): p 158-166, 2005, KR2009-0121694A), OKT-3 antibody (Experimental Hematol., 29(1): p 104-113, 2001) which stimulates CD3, and LPS (J. Immunol., 165(1): p 139-147, 2000). However, such studies have merely found a modification of the use of IL-2 and a new proliferator, but did not suggest an epochal method for proliferation. It is generally known that, when NK cells are cultured using IL-2 or other cytokines or chemicals, the number of NK cells is increased only by about 3-10 times the initial number of NK cells.
Some researchers reported that NK cells were expanded using cancer cell lines as feeder cells. It was reported that the use of the leukemia cell line CTV-1 showed little or no improvement in proliferation (J. Immunol., 178(1): p 85-94, 2007) and that culture using EBV-LCL for 21 days increased the cell number by an average of about 490 folds (Cytotherapy, 11(3): p 341-355, 2009). Also, culture of NK cells for 3 weeks using artificial APC (antigen-presenting cell) obtained by expressing 4-1BBL and membrane-bound IL-15 in the K562 cells increased the NK cell number by an average of 227 folds, and high cytotoxicity appeared in vitro and in vivo, but limited proliferation caused by cell death was shown (Cancer Res., 69(9): p 4010-4017, 2009). Recently, there was a reported that culture of NK cells for 3 weeks in the K562 cell line transfected with MICA, 4-1BBL and IL-15 increased the NK cell number by an average of 350 folds (Tissue Antigens, 76(6): p 467-475, 2010), and there was a report that, when NK cells were cultured for 2 weeks using the K562 cell line transfected with membrane-bound IL-21 while they were stimulated at 7-day intervals, the cell number was increased by an average of 21,000 folds. However, the methods unsuitable for guaranteeing safety important for clinical application were used, because all cancer cell lines were used. In addition, because specific cancer cells are used as feeder cells, the resulting NK cells have priming specificity for the specific cancer cells.
Cells obtained by enrichment of NK cells from peripheral blood leukocytes (PBLs) without the isolation of NK cells have low cytotoxicity compared to pure NK cells, and contain T-cells that recognize self and non-self cells by autologous MHC molecules. Thus, the cells are limited to autologous transplantation, as long as T-cells are not removed. Recently, there have been developed a method comprising NK cells isolation step, and expanding the isolated NK cells with suitable stimulation using feeder cells, and a method of selectively expanding NK cells using whole PBL or peripheral blood mononuclear cells (PBMCs). In addition, there was reported a method for culturing NK cells, which comprises a process of culturing NK cells using a medium containing anti-CD3 antibody and interleukin protein in the presence of peripheral blood leukocytes (KR 10-2010-0011586 A).
The general expansion process for allogeneic application starts with two sequential steps of magnetic depletion of CD3+ T cells and enrichment of CD56+ NK cells. In order to stimulate NK cell proliferation, irradiated feeder cells such as PBMCs [Cytotherapy 12: 750-763, 2010], Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) [Cytotherapy 11: 341-55, 2009] are often used. Irradiated feeder cells stimulate NK cells through both humoral factors and direct cell-to-cell contact [Blood 80: 2221-2229, 1992].
In the present invention, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy volunteers for clinical use. After a single step of magnetic depletion of CD3+ T cells, the depleted PBMCs were stimulated and expanded with irradiated autologous PBMCs repeatedly in the presence of OKT3 and IL-2, resulting in a highly pure population of CD3-CD16+CD56+ NK cells which is desired for allogeneic purpose.
Meanwhile, albumin is one of proteins constituting the basic substance of cell and has the lowest molecular weight among simple proteins present in nature. Albumin is generally known to be used as a preservative for biological formulations, but the use of albumin to increase the stability of NK cells has not yet been reported.
As described above, a variety of methods for culturing NK cells have been described, but there is still an urgent need for a technology which can safely and stably culture and expanding a sufficient number of NK cells for application to immune cell therapy using a clinically friendly method and allows the produced NK cells to be stably stored for a long period of time and to be supplied when required. Particularly, in order to use living NK cells for therapeutic purposes, it is required to overcome the shortcoming of the NK cells in that the period in which the activity thereof is maintained (that is, availability period) is only several days. Thus, there is an urgent demand for a technology which can significantly improve the utilization of immune cells by culturing and producing NK cells, freeze-storing the produced NK cells, and thawing and providing the stored cells when required.